Broad Long-Term Objectives: Develop and apply tools for the determination of biologically active peptides at physiologically relevant concentrations and volumes of sample. Specific Aims: Technical Advance: Establish a novel detection method for the liquid chromatographic determination of peptides that is peptide selective-only peptides give peaks, and sensitive on a concentration basic-picomolar. Such peptides as oxytocin, vasopressin, neuropeptide Y, substance P, delta sleep-inducing peptide, enkephalins, and others will be measurable without immunoassay at physiological levels. Biological Problem: Through the application of this new and powerful method, we will work to understand the mechanisms of excitotoxicity-the events in which highly stimulated nerve cells die. Methods: Technical: We will develop a novel detection technique with the selectivity of fluorescence. Biological: Models of ischemia: in vitro: oxygen starvation of rat hippocampus slices. in vivo: clamping of carotid arteries of adult rats; hypoxia/ishemia in neonatal rats. Sampling: in vitro, perfusion; in vivo, microdialysis, also tissue extraction. Health Relatedness: What the proposed procedure offers over traditional immunoassays is the ability to see the whole peptide picture, including what is happening to peptide precursors, and how active peptides are metabolized. This is because the analysis does not rely upon specific biological binding reactions. In studies of virtually any regulatory system, such information will be useful in understanding pathology. Our specific application is to the role of peptides on the glutamatergic system in the rat brain, and in particular trying to understand the phenomena that lead to nerve cell death in such incidents as stroke, head trauma, epilepsy, deep hypoglycemia, and several syndromes that include dementias such as AIDS-related dementia. Current knowledge allows us to say that it is possible in principle to minimize cell death, and the related practical consequences, resulting from exitotoxicity, but it does not allow us to say how. A more detailed understanding of the mechanisms leading to cell death, including the roles of peptides, will allow evaluation of drug strategies against nerve damage.